cell cycle 6: apoptosis Flashcards
why do you need programmed cell death?
to kill:
- harmful cells (eg cells with viral infection / dna damage)
- developmentally defective cells (eg b lymphocytes with self antibodies)
- excess / unneccessary cells (eg during embryonic development)
- obsolete cells (eg mammary epithelium at end of lactation)
- exploitation (chemotherapeutic killing of cells)
what is necrosis?
unregulated cell death associated with trauma, cellular disruption and an inflammatory response
what is apoptosis?
programmed cell death NOT associated with inflammatory response
- controlled disassembly of cellular contents without disruption
what happens during necrosis?
plasma membrane becomes more permeable -> cell swelling -> rupturing of cellular membranes -> release of proteases -> autodigestion & dissolution of cell -> localised inflammation
what happens during apoptosis?
latent phase: death pathways activated but cells appear morphologically the same
execution phase: loss of microvilli & intercellular junctions
- cell shrinkage
- loss of plasma membrane asymmetry (phosphatidylserine appears in outer leaflet)
- chromatin + nuclear condensation
- dna fragmentation
- formation of membrane blebs
- fragmentation into membrane-enclosed apoptotic bodies
how can DNA modification during apoptosis be measured?
dna in agarose gel separated by size -> dna ‘ladders’ formed
what is a tunel assay?
fluorescent tags added to ‘ends’ caused by dna fragmentation
- more ends seen in fragmentation -> more fluorescence
what are caspases?
cysteine-dependent aspartate-directed proteases
- executioners of apoptosis
- activated by proteolysis
- cascade of activation
what are the 2 subclasses of caspases?
initiator caspases (2,8,9&10) effector caspases (3,6&7)
initiator caspases:
have CARD end terminal (caspase recruitment domain)
- have p20 & p10 domains
- 8&10 also have DED (death effector domain) before p20&10 effector caspases
- homotypic (dimerise with same type of caspase eg 8+8)
what is caspase maturation?
caspases synthesised as procaspases (with prodomain, LS & SS domain) -> prodomains cleaved -> folding of 2 large and 2 small domains to from large hetero-tetramer (activated by heterodimerisation) -> initiates caspase cascades
outline the caspase cascades:
8 & 9 -> (7) + 3 -> (2 & 1) + 6 -> 10
what do effector caspases do?
- execute apoptotic programme
- cleave and inactivate proteins / complexes (eg nuclear lamins -> nuclear breakdown)
- activate enzymes (incl protein kinases, nucleases) by direct cleavage or cleavage of inhibitory molecules
what are the mechanisms of caspase activation?
death by design (receptor mediated pathways - extrinsic)
death by default (mitochondrial death pathway - intrinsic)
what are death receptors?
trimeric transmembrane ligands
- all have death domain (DD)
- signal by connecting with adaptor proteins which recruit other proteins on site
what are the adapter proteins?
proteins needed to trigger intracellular effects (in addition to the ligand + death receptor)
- FADD (DED + DD) & FLIP (DED)
- FADD activates & FLIP deactivates
- DD & DED bind to similar domains on other proteins
what happens during the receptor mediated pathway?
Fas binds to Fas-L (on plasma membrane of CTLs)
-> ligand brings together three death receptor monomers to form trimer
-> DD of adapter protein (FADD) binds DD of death receptor (Fas)
-> DED of adaptor protein binds DED on procaspase 8 (in this example)
=> this whole complex is called the DISC (death-inducing signalling complex)
-> three procaspases that have been brought together cross-cleave subunits to form their active hetero-tetramer
what is FLIP?
procaspase analogue
- only has 2 DED domains and no proteolytic activity -> competes with procaspase 8 for DISC binding sites -> reduces caspase activation
what happens during the mitochondrial pathway?
cellular stress causes damage to mitochondrial membrane
- > loss of mitochondrial membrane potential (ΔΨ)
- > membrane becomes leaky
- > cytochrome C released into cytoplasm
- > stimulates formation of apoptosome complex
- > > > caspase released from complex
what is the apoptosome?
heptamer made up of 7 Apaf-1 profeins (with CARD & WD40 domain)
what happens when cytochrome C binds to the apoptosome?
7 apaf1 molecules assemble into wheel-like heptamer around their CARD domains
- > CARD domains on 7 procaspase 9 molecules bind to apoptosome
- > proximity of procaspases allows cross-cleavage & activation to caspase 9 (similar to in DISC of receptor-mediated pathway)
how do energy levels in the cell determine apoptosis v necrosis?
apoptosome requires lots of ATP
- so cells with lots of atp will favour apoptosis
what proteins modulate apoptosis?
Bcl-2
- all contain BH3 domain
what are the 2 types of Bcl-2 proteins?
anti-apoptotic: associated with mitochrondrial membrane (eg Bcl-2 itself)
pro-apoptotic: in cytosol initially then move to mitochondria (eg Bid)
what is Bid?
Bcl-2 -> vleaved by caspase 8 of extrinsic pathway -> inserts into the mitochondrial membrane to disrupt ΔΨ -» activates intrinsic pathway too
what is Bad?
pro-apoptotic Bcl-2 that usually isn’t free to act
how is Bad phosphorylated?
growth factor binds to RPTK at cell membrane
- > PI3K (lipid kinase with adapter protein) binds to phosphorylated tyrosin on RPTK
- > PI3K phosphorylates PIP2 to PIP3
- > PIP3 acts as docking site for PKB / Akt
- > PKB/Akt phosphorylates Bad
- > complexes with 14-3-3 (a protein that just holds Bad -> on mitochondrial membrane Bcl-2 is bound to Bax (via their BH3 domains) however if growth factor is absent Bad is not phosphorylated
- > displaces Bax from Bcl-2
- > Bax molecules associate with each other
- > form pore in mitochondrial membrane
- > leakiness
- > apoptosis
what is PKB/Akt?
proto-oncogene
- inactivates Bad
- inactivates caspase 9
- inactivates pro-apoptotic FOXO transcription factors
- stimulates protein synthesis
what is PTEN?
phosphatase that hydrolyses PIP3 -> PIP2 -> therefore PTEN affects how well PKB/Akt pathway inactivates Bad
what are IAPs?
inhibitors of apoptosis proteins
- bind to procaspases & caspases and inhibit them -> prevents apoptosis
